IRMS Sample Introduction System and Method

ABSTRACT

A sample introduction system for a spectrometer comprises a desolvation region that receives or generates sample ions from a solvent matrix and removes at least some of the solvent matrix from the sample ions. A separation chamber downstream of the desolvation region has a separation chamber inlet communicating with the desolvation region, for receiving the desolvated sample ions along with non-ionised solvent and solvent ion vapours. The separation chamber has electrodes for generating an electric field within the separation chamber, defining a first flow path for sample ions between the separation chamber inlet and a separation chamber outlet. Unwanted solvent ions and non-ionised solvent vapours are directed away from the separation chamber outlet. The sample introduction system has a reaction chamber with an inlet communicating with the separation chamber outlet, for receiving the sample ions from the separation chamber and for decomposing the received ions into smaller products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 16/575,312, filed Sep. 18, 2019, which is acontinuation of U.S. Ser. No. 16/130,760, filed Sep. 13, 2018, now U.S.Pat. No. 10,446,380, which is a continuation of U.S. Ser. No.15/402,108, filed Jan. 9, 2017, now U.S. Pat. No. 10,090,140, whichclaims priority to British patent application serial no. 1600569.6,filed Jan. 12, 2016. The contents of each application are herebyincorporated by reference herein in its entirety for all purposes.

FIELD OF THE INVENTION

This invention relates to a sample introduction system for an isotoperatio mass spectrometer or isotope ratio optical spectrometer, and amethod for coupling such an isotope ratio spectrometer (IRS) to a supplyof sample entrained with a matrix/solvent.

BACKGROUND TO THE INVENTION

Isotope ratio mass spectrometry is a technique which accurately andprecisely measures variations in the relative abundances of isotopes,i.e. isotopic ratios, of elements such as ¹³C/¹²C, ¹⁸O/¹⁶O, ¹⁶N/¹⁴N and³⁴S/³²S in molecules.

Prior to analysis, a sample typically undergoes oxidation, pyrolysis orreduction at an elevated temperature to produce gases of molecules, forexample, CO_(x), NO_(x), H₂O. The gases are then introduced into the IRSfor isotopic analysis. In the isotope ratio spectrometer (IRS), thegases are ionised and the ratios of corresponding isotopes are measuredfor example by comparing outputs of different collectors. The ratios ofthe isotopes of interest are typically measured relative to an isotopicstandard in order to eliminate any bias or systematic error in themeasurements.

For isotopic analysis of specific compounds within a complex mixture, itis desirable to perform a separation prior to the isotopic analysis.Currently, this separation is performed by gas chromatography, which canbe coupled to an IRMS using a combustion oven.

Liquid chromatography (LC) is an established technique in the field ofbiochemistry and pharmacology. However, coupling an IRS to a liquidchromatography system presents technical challenges because LC mobilephase is usually organic and therefore produces the same products assample molecules of interest, thus interfering with the isotopicanalysis. There have been various attempts at coupling liquidchromatography to IRMS, as identified below.

“Moving-wire device for Carbon Isotopic Analyses of Nanogram Quantitiesof Nonvolatile Organic Carbon” (A. L. Sessions, S. P. Sylva and J. M.Hayes, Anal. Chem., 2005, 77, 6519-6527) describes a method foranalysing ¹³C ratios of involatile organic samples dissolved insolution. The output solution of the separation system is dried onto anickel wire to remove the mobile phase from the sample. The residualsample is then combusted and the evolved CO₂ is analysed by IRMS.However, both the precision and sensitivity of this method are limitedby a high background level of CO₂ derived from carbon within the wire.

Another method of coupling a liquid chromatography system to an IRMS ispresented in ““Continuous-Flow Isotope Ratio Mass Spectrometry Using theChemical Reaction Interface with Either Gas or Liquid ChromatographyIntroduction” (Y. Teffera, J. Kusmierz, F. Abramson, Anal. Chem., 1996,68, 1888-1894)”. In this method, the solution exiting from the liquidchromatography system undergoes desolvation at semi-permeable membranesprior to chemical oxidation of the dry aerosol. The oxidised productsare then analysed by IRMS. However, the method described does not removethe mobile phase to the required ultra-low levels of solvent, forexample, to a solvent/sample ratio better than 1:100.

Wet chemical oxidation (LC-Isolink™) addresses the problem of bothearlier methods and allows coupling to liquid chromatography. Thesolution output from the chromatography system is mixed with anoxidizing agent and supplied to an oxidation reactor. In the oxidationreactor the organic compounds are converted into CO₂, which is thenanalysed in the IRMS. However, there is no separation of the mobilephase from the sample and therefore, this method is not suitable forseparation methods that require an organic mobile phase.

In the fields of pharmaceutical and life sciences, the typical sampleincludes organic molecules dissolved in an organic solvent. For suchsamples, separation of the molecules from the solvent is generallycarried out with an organic mobile phase using techniques such as highperformance liquid chromatography, capillary-zone electrophoresis andsize-exclusion chromatography. As a result, the output of the separationapparatus also consists of an organic sample dissolved in an organicsolvent.

The presence of this organic solvent would result in production of alarge amount of CO₂ during combustion and hence an extremely highbackground CO₂ in the spectrum produced by IRMS.

In order for analysis by IRMS of an organic molecule dissolved in anorganic solvent, a great reduction in the solvent/sample ratio from100-1,000,000:1 to less than 1:100-1000 i.e. a reduction of 5-8 ordersof magnitude or higher is required.

None of the existing techniques, as identified above, can reduce theorganic solvent/organic sample ratio to the required ultra-low levels.

Therefore, a sample introduction system which can couple a supply ofsample entrained with any solvent to an IRMS is required.

The present invention seeks to address this problem by providing a newapproach to separation of sample molecules from more volatile moleculesof the mobile phase.

SUMMARY OF THE INVENTION

According to a first aspect of this invention, a sample introductionsystem for an IRMS is provided.

As noted above, the challenge faced by current techniques for analysinga sample dissolved in a solvent, is how to reduce the organicsolvent/organic sample ratio to the required ultra-low levels so thatthe solvent does not contribute significantly to the recorded spectrum,e.g. isotopic spectrum. In this way, an improved quantitation ofisotopic ratios can be achieved.

The sample introduction system of the claimed invention requiresionization of the sample prior to decomposition, preferably in a sprayionization source. The ionised sample is then desolvated. The resultingdesolvated ions are then preferably moved, optionally accelerated, in aseparation chamber in first direction by an electric field whilst beingmoved in a second, different direction by, for example, a flow of gas,or an electric or magnetic field (static and/or varying). The result isthat sample ions of a desired species having a particular mobilityand/or mass to charge ratio (or range of mobilities and/or mass tocharge ratios) are directed to an outlet of the separation chamber, foronward reaction/combustion/pyrolysis/reduction, whilst unwanted solventions and uncharged solvent molecules are forced either to move along adifferent path, or randomly/indiscriminately in multiple directions, sothat, in either event, they do not pass out of the separation chamberfor downstream analysis, and are instead swept away or lost.

The sample introduction system of the claimed invention differs fromthat employed in a typical IRS, in that it requires ionization of thesample prior to decomposition (by an ionisation source within the sampleintroduction system). The ionization takes place upstream of theseparation chamber, which can then act to separate the sample fromsolvent ions and solvent vapour as described. The sample ions exitingthe separation chamber into the reaction chamber are then decomposedtherein to smaller products, typically molecular products (e.g.including one or more of CON, NON, H₂O in the case of acombustion/oxidation chamber, x being typically 1 or 2) and theresultant decomposed products are analysed. In IRMS, this implies afurther ionisation source to ionize the resultant decomposed products topermit subsequent analysis. In IR optical spectroscopy, isotope ratiosof the resultant decomposed products may be determined fromspectroscopic measurements in a cavity. For example, infra-redwavelengths corresponding to the greatest optical absorption of theproducts may be determined.

The pressure in the separation region is desirably lower than thepressure in the desolvation region, so that ions are drawn from thedesolvation region and into the separation region as a jet. The geometryof the aperture or channel between the desolvation and separationregions may also be configured to improve transfer of ions into theseparation region as known in the prior art. The sample introductionsystem preferably operates at around atmospheric pressure. For example,the desolvation region may be held at atmospheric pressure (100 kPa)whilst the separation chamber may be evacuated to a pressure preferablynot more than half the pressure in the desolvation region, e.g. around10-30 kPa (0.1-0.3 bar). Alternatively, the separation chamber may beheld at around atmospheric pressure, with then the pressure in thedesolvation chamber being raised to preferably at least twice thatpressure, e.g. around 200-300 kPa (2-3 bar). Such a large difference inpressures is preferable because it creates a supersonic jet followed byshock waves, thus accelerating gas transfer between regions. This inturn reduces the dependency on sample and conditions (e.g. humidity) inthe desolvation region.

Thus preferred embodiments of the present invention provide a way ofremoving large quantities of solvent at a relatively high pressure(preferably atmospheric pressure). Providing a way of removing solventat relatively high pressure is desirable, since it provides increasedefficiency and reduces sample losses. It also permits coupling to thereaction chamber, for decomposition of the sample ions. A pressurearound atmospheric pressure in the separation chamber and higher in thedesolvation region is thus most preferable.

The invention also extends to an IRMS or IROS having such a sampleintroduction system.

In addition to a sample introduction system, the present invention alsoextends to a method of introducing a sample into an Isotope Ratio MassSpectrometer.

Further aspects of the present invention provide a sample introductionsystem for an IRMS, comprising a first ionization source arranged toreceive a sample from a liquid sample preparation region and to ionizethe received sample to produce sample ions in a solvent matrix, adesolvation region to remove at least a proportion of the solvent matrixfrom the sample ions, a separation chamber positioned downstream of thedesolvation region for receiving the desolvated sample ions along withsolvent vapours comprising non-ionised solvent and solvent ions, andseparating out sample ions of interest for further analysis; a reactionchamber arranged to receive the separated sample ions of interest and toreact the said sample ions of interest to produce products; and a secondionization source to ionize the products of the reaction chamber so asto produce product ions for analysis by an IRMS.

A method of sample introduction is also contemplated, which comprisesreceiving a sample from a liquid sample preparation region, ionizing, ina first ionization source, the received sample to produce sample ions ina solvent matrix, removing at least a proportion of the solvent matrixfrom the sample ions in a desolvation region, receiving, from thedesolvation region, the desolvated sample ions along with solventvapours comprising non-ionised solvent and solvent ions, and separatingout sample ions of interest for further analysis in a separation chamberpositioned downstream of the desolvation region; receiving the separatedsample ions of interest in a reaction chamber, and reacting the saidsample ions of interest to produce products; and ionizing, in a secondionization source, the products of the reaction chamber so as to produceproduct ions for analysis by an IRMS. That method may then also includemass analysing the product ions in an IRMS device.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be put into practice in a number of ways and somespecific embodiments will now be described by way of example only andwith reference to the accompanying drawings in which:

FIG. 1 shows a schematic view of a system comprising a liquid samplepreparation region and a sample introduction system coupled to anisotope ratio mass spectrometer (IRMS);

FIG. 2 shows, in further detail, the liquid sample preparation regionand sample introduction system of FIG. 1, the sample introduction systembeing in accordance with a first exemplary embodiment of the presentinvention;

FIG. 3 shows, also in further detail, the liquid sample preparationregion and sample introduction system of FIG. 1, the sample introductionsystem being in accordance with a second exemplary embodiment of thepresent invention;

FIGS. 4a and 4b show schematic arrangements of alternate sample reactionarrangements suitable for the sample introduction systems of FIGS. 1-3;

FIG. 5 shows a schematic arrangement of a part of the liquid samplepreparation region and sample introduction system of FIGS. 1, 2 and 3,coupled with an orbital trapping mass spectrometer.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

FIG. 1 shows a schematic diagram of a system 1 comprising a liquidsample preparation region 10 and a sample introduction system 50 coupledto an isotope ratio mass spectrometer (IRMS) 100, having a detector 150.

The liquid sample preparation region 10 of the system 1 has anautosampler 20 for providing a sample to an injector 25 where the sampleis entrained with a pumped liquid mobile phase. Embodiments of thepresent invention are particularly concerned with analysis of apharmaceutical or life sciences sample, which typically contains largeorganic molecules dissolved in a liquid solvent which is a mixture ofacidified water and organic solvent such as acetonitrile or methanol invarying ratios.

The sample entrained with the liquid mobile phase is provided to aliquid separator 30 by means of a pump 40. Any liquid separator 30 maybe employed to separate a component or components of interest in theliquid sample, for example, capillary zone electrophoresis (CZE), highperformance liquid chromatography (HPLC) or size exclusionchromatography (SEC) column. The liquid separator typically separatesone or more components of the sample in the solvent matrix so that theyelute from the liquid separator separated in time. The structure of theliquid separator as known in the art is not discussed in further detailhere.

The output of the liquid separator (eluate) comprises the separatedsample entrained in solvent. The output of the liquid separator 30 isfluidically coupled to an inlet of a sample introduction system 50.

The sample introduction 50 comprises a desolvation chamber 60 includinga first ionization source 65. Various types of first ionization source65 may be employed, such as a nanospray ionization source, thermosprayionization source, atmospheric pressure chemical ionization source,atmospheric pressure photo-ionization source, glow discharge orlow-temperature plasma source, inlet ionization source etc. The firstionization source 65 receives the sample entrained with solvent,preferentially ionizes the sample, and evaporates the solvent from thesample in the desolvation chamber 60 so as to produce desolvated sampleions and solvent vapours. The solvent vapours comprise non-ionisedsolvent molecules and/or solvent ions. High efficiency of conversion ofthe sample into ions is important for good sensitivity of the method.

A separation chamber 70 downstream of the desolvation chamber 60receives the desolvated sample ions and solvent vapours, via aseparation chamber inlet 72. Within the separation chamber 70, thedesolvated sample ions and solvent vapours experience an electric field(E) that moves or accelerates the desolvated ions from the entrancetowards the exit of the chamber. Via a gas flow in a different directionto the electric field, via crossed electric and magnetic fields, via acombination of static and varying electric fields, or otherwise, sampleions of a selected mobility (or selected range of mobilities) are thendirected towards a separation chamber outlet 75. This process will bedescribed in further detail below, in connection with FIGS. 2 and 3.

The selected ions exiting the separation chamber outlet 75 enter areaction or decomposition chamber. In FIG. 1, the reaction ordecomposition chamber is a reaction chamber 80, positioned downstream ofthe separation chamber 70. In the reaction chamber 80, the ions of theselected species are decomposed at elevated temperatures, optionally inthe presence of a catalyst, into a combination of light gases such asCO₂, NO_(x), H₂O and H₂.

A CO₂ separation unit 90 of the sample introduction system 50 isoptionally positioned downstream of the reaction chamber 80 forselective removal of the CO₂ from the combusted sample in known manner.The CO₂ separation unit 90 comprises a membrane exchanger of planargeometry configured to separate CO₂ from the remaining gases. A flow ofhelium gas is provided in a direction normal to the plane of themembrane. In that case, the CO₂ gas is then carried in the flow ofhelium and may be dried using a dryer 95 (eg Nafion™). The CO₂separation unit 90 is advantageous if the analysis to be performed byIRMS is specifically of CO₂.

The resulting gases (e.g. CO₂, NO_(x), H₂, H₂O) then leave the sampleintroduction system 50 and enter the IRMS 100 either directly or via anopen split. The IRMS may be any suitable known device, eg the Delta VIMIRMS manufactured and sold by Thermo Fisher Scientific, Inc.Alternatively, an optical based isotope ratio spectrometer (e.g. ThermoScientific Delta Ray™) may be employed, for analysis of ¹³O/¹²O, ²H/¹Hor ¹⁸O/¹⁶O isotopic ratios etc.

Merely by way of example, therefore, FIG. 1 shows a first part of theIRMS 100 which comprises a second ionization source 110. The secondionization source 110 may, for example, be an inductively coupled plasma(ICP) ionization source or, where higher sensitivity is desirable, anelectron impact (EI) ionization source, or the like. Ions generated bythe second ionization source 110 are accelerated and then passed throughan entrance slit 120 which controls the ions entering the IRMS 100, anddetermines the IRMS 100 resolution.

The accelerated ions then enter an optional electric sector 130, a setof ion optics 140, and a magnetic sector 145. Ions are thus separated inaccordance with their mass to charge ratio and arrive at a detectorarrangement 150 positioned at the focal plane of the ion beam. Thedetector arrangement 150 contains a detector 160 which may, for example,be a multiple collector arrangement under the control of a controller170. The controller may also comprise a data acquisition system. Asingle detector may alternatively be used e.g. with scanning of the ionmass-to-charge ratio by the magnetic sector.

The details of the ion separation in the liquid phase and detectiondownstream of the sample introduction system 50 do not form a part ofthe present invention and so will not be described in further detail. Itwill moreover be understood that various types of IRMS spectrometer maybe employed, such as continuous flow and duel-inlet IRMS.

Having described, in general terms, the stages of ionization,separation, decomposition and detection of sample ions of interest, themanner by which pyrolized or combusted or reduced sample ions can beintroduced to the IRMS, whilst solvent molecules are removed, will nowbe described with reference to FIGS. 2 and 3. FIG. 2 shows a moredetailed schematic diagram of a sample introduction system 50, intowhich an eluate is introduced from a liquid sample preparation region10.

As explained above in connection with FIG. 1, the sample injector 20injects a sample entrained with liquid mobile phase into the liquidseparator 30 by a pump 40. The eluate output from the liquid separator30 then enters desolvation chamber 60. It is to be noted that, in anyliquid separation, the ¹³O or ²H isotope-containing molecules mightelute slightly differently from the base-isotope molecule, leading tonoticeable fractionation. It is desirable to correct for any suchfractionation.

Upon entering the desolvation chamber 60, the eluent from the liquidsample preparation region 10 is converted into charged droplets andthen, after desolvation of the droplets, ions by the first ionizationsource 65 which is, as noted above, preferably a spray ionizationsource. The resultant ions travel across the desolvation chamber towardsa heated channel 200 which guides sample ions towards an outlet of thedesolvation chamber 60.

It is preferable that the arrangement of FIG. 2 employs a planargeometry, that is, the sectional view shown in FIG. 2 extendsorthogonally to the plane of the drawing. The heated channel 200 may be,for example, between 0.8 and 1 mm high and 5-10 mm wide and between 20and 100 mm in length, which is heated up to 500-700° C. The heatedchannel dimensions define the opening of the separation chamber inlet72. Such an opening allows transmission of between 10 and 100 nA of ioncurrent into the separation chamber 70.

In addition to the heated channel 200, a flow of heated gas mayadditionally or alternatively be supplied to the desolvation chamber 60.Both the heated channel 200 and the heated gas flow may significantlyimprove the degree of desolvation of the ionized eluent entering thedesolvation chamber 60.

The pressure in the desolvation chamber 60, P_(sampling), may be greaterthan, the same as or less than atmospheric pressure P₀. The relativepressures in the various parts of the sample introduction system 50 mayassist in the removal of unwanted solvent prior to injection into theIRMS 100.

In order to achieve efficient and rapid transfer, it is preferable thatthe pressure in the separation chamber 70 is lower than the pressure inthe desolvation chamber 60. In particular, it is preferable to form ajet leaving the aperture 72 between the desolvation chamber 60 andseparation chamber 70, with P_(sampling)>2*P_(sep), where P_(sep) is thepressure within the separation chamber 70. For example, P_(sampling) maybe between 200-300 kPa (2 to 3 bar), whilst P_(sep) equals P₀ (that is,the separation chamber is held at atmospheric pressure). Alternatively,P_(sampling) equals P₀—i.e. the desolvation chamber 60 is held atatmospheric pressure, whilst in that case P_(sep) equals 10-30 kPa(0.1-0.3 bar). One or more pumps (not shown in FIG. 2) may be providedin order to adjust the pressure within the separation chamber 70 and/ordesolvation chamber 60, above or below atmospheric pressure.

The separation chamber 70 of FIG. 2 separates ions of different species,and removes unwanted neutral solvent molecules, using a technique knownas differential mobility analysis (DMA). The general principles of thetechnique are described in, for example, U.S. Pat. No. 5,869,831.

The separation chamber 70 of FIG. 2 comprises first and second generallyplanar electrodes 210, 220 which are located in opposition to oneanother across a separation gap in the longitudinal direction of thesample introduction system 50. The first and second electrodes 210, 220are separated by a gap in the range of 10-50 mm with gas blown in by afan or sucked away by a pump. The separation chamber inlet 72 is formedin or through the electrode 210 whilst the separation chamber outlet 75is formed in or through the electrode 220. Each aperture is, preferably,slit shaped (e.g. of the dimensions noted above for the slit shapedheated channel 200).

The voltages applied to each of the electrodes 210, 220 are selected onthe basis of the sample ions of interest in the sample. As shown in FIG.2, in the specific example provided, the first electrode 210 has avoltage of 1000 volts applied to it whilst the second electrode 220 hasa voltage of 200 volts applied to it. For pressures in the range 10-1000Pa (0.1-10 mbar), it is preferable not to exceed 300V between theelectrodes 210 and 220, in order to avoid gas discharge. Ions ofdifferent species A, B and C have different mass to charge ratios andare accordingly accelerated to different drift velocities within theseparation chamber 70.

The desolvated sample ions and solvent vapours enter the separationchamber 70 via inlet 72 as a jet, in a direction X as shown in FIG. 2. Adry, ion free inorganic gas, preferably of high or ultra-high purity, issupplied in a direction Y transverse to the direction X (in other words,transverse to the longitudinal axis of the sample introduction system50). In FIG. 2, the dry gas is introduced in a direction Y that isperpendicular to the direction X. However it will of course beunderstood that the dry gas may be introduced at any suitable anglerelative to the direction X, provided only that the dry gas flowdirection intersects the direction of flow of ions as they enter theseparation chamber 70 and are accelerated by the DC electric field.

The separation chamber inlet 72 is offset from the separation chamberoutlet 75 in the Y direction. The combination of the DC electric fieldaccelerating ions in the direction X and the dry gas flow imparting acomponent of movement to the ions in the direction Y, is that ionsdescribe flow paths having both an X and a Y component as they travelacross the separation chamber 70. Ions of different species havedifferent masses and collisional cross sections, so that the interactionbetween molecules of the dry gas and ions within the separation chamber70 will differ between ion species in the separation chamber 70. Inother words, ions of a first species A having a first electricalmobility (first mass and collisional cross section), will be deflectedalong a first path. Ions of second and third species B, C, havingrespective second and third electrical mobilities (mass/collision crosssections), however, will be deflected respectively along second andthird paths, each different to one another and to the first path. In theexample shown in FIG. 2, the particular combination of applied voltagesand gas flow chosen, results in the deflection of ions of species B (andonly these) into the separation chamber outlet 75. Generally speakingthe direction of travel of wanted sample ions entering the reactionchamber 80 is in the X direction, i.e. parallel with the direction offlow of ions in the jet entering the separation chamber 70, but shiftedin the Y direction.

In this manner, dust and unwanted neutral and charged solvent molecules,which typically form as large clusters with high mass to charge ratiosand high collisional cross sections, can be separated from wanted sampleions via the separation chamber 70, because the solvent clusters havetoo high a collision cross section to follow the trajectories of thesample ions. Moreover, neutral solvent molecules entering the separationchamber 70 will not be accelerated by the electric field towards theelectrode 220, and so will also be swept away by the flow of dry gas.

If the end goal of the sample analysis is to study C or O isotopicratios, then the dry gas may be, for example, argon, nitrogen or thelike. For analysis of N isotopes, argon or oxygen might instead beemployed. The sample introduction system 50 is, of course, not limitedonly to such elemental isotopes, and could equally be employed to studyisotopic ratios of CO₂, H₂/HD for pharmaceutical and life sciences, andso forth.

As a result of the markedly different electrical mobilities of thesample and solvent ions, typically a very low resolving power ofseparation (perhaps 2-3) is sufficient to separate the sample andsolvent ions. Appropriate resolving power is defined by selectingappropriate geometrical and electrical parameters of the separator. Sucha very low resolving power of separation results in a uniformtransmission of sample molecules of broad mass range and negligibleisotopic discrimination. Calibration compounds can be employed togenerate correction coefficients to take into account efficiency ofionization. The strong electric field is created by a voltage dropbetween the electrodes 210 and 220 and the optional heated channel 200at the exit of the desolvation region 60 may permit complete desolvationof the sample ions.

In the embodiment of FIG. 2, the reaction chamber 80 is coupled to theseparation chamber outlet 75 via a reactor inlet 265. At this interface,it is desirable to provide a counter flow of dry inorganic gas so thatany solvent molecules that still remain entrained with the wanted sampleions upon arrival at the separation chamber outlet 75 are prevented fromentering the reaction chamber 80. To achieve this, it is desirable thatthe pressure Pr within the reaction chamber 80 (which couldalternatively be a pyrolysis or reduction chamber) is higher than thepressure in the separation chamber 70 (i.e. P_(r)>P_(sep)). Then, the DCelectric field in the separation chamber 70 applies a force in thepositive X direction to wanted sample ions to drive them into thereaction chamber 80, whilst the counterflow of gas is drawn into theseparation chamber 70 from the direction of the reaction chamber 80 bythe pressure difference in the negative X direction.

The reaction chamber 80 is preferably a non-porous aluminium tube thatcontains three separate twisted wires made of copper, nickel andplatinum and is typically maintained at 1030 degrees Celsius. This typeof reaction chamber is described inhttp://stableisotopefacility.ucdavis.edu/ASITA/Eby-presentation1.pdf.

FIG. 3 shows an alternative arrangement of a sample introduction system60′, into which an eluate is introduced from the liquid samplepreparation region 10. Those components which are common to FIGS. 2 and3 are labelled with like reference numerals. Where the function of thecommon parts is the same as between FIGS. 2 and 3, this will beindicated below to avoid repetition.

In FIG. 3, an eluate is generated by a liquid separator 30 fed from asample injector 20 by a pump 40. The eluate then enters a firstionization source 65 forming an upstream part of a desolvation region60′. The first ionization source 65 may be of any of the same types asdescribed above in respect of FIG. 2. The desolvation region 60′ is, inthe exemplary embodiment of FIG. 3, not sealed against outer atmosphere.The pressure P_(sampling) in the desolvation region 60′ is thusatmospheric (P_(sampling)=P₀).

Ions generated by the first ionization source 65 traverse a gap andarrive at a heated channel 200, whose function and configuration may beas previously described. From there, desolvated ions and remainingsolvent vapours enter a separation chamber 70′. The separation chamberhas an inlet 72 through which the heated channel 200 extends, so thatthe heated channel directs the desolvated ions and solvent vapours intothe separation chamber 70′ in a direction generally parallel with the Xdirection shown in FIG. 3.

Extending in the +/−X direction is a first DC electrode 300. An apertureplate 310 is separated from the first DC electrode in the Y direction,and a separation chamber outlet 75 is formed in that aperture plate 310.A power supply (not shown) applies a potential difference ofsubstantially constant voltage between the first DC electrode 300 andthe aperture plate 310; for example the aperture plate 310 may begrounded whilst a potential of 300V is applied to the first DC electrode300. Such a potential difference results in a DC electric field beinggenerated in the separation chamber 70′. The separation chamber inlet 72is positioned between the first DC electrode 300 and the aperture plate310, so that ions entering the separation chamber 70′ as a jet in thedirection X experience a force in the Y direction. The combination ofthe velocity of the ions in the jet that enters the separation chamber70′ (in the direction X), and the electric field that imparts a force inthe direction Y, causes ions to commence a curved trajectory.

Extending in the +/−Y direction to either side of the separation chamber70′ are first and second combined AC/DC electrode stacks 320,330. Thepower supply is configured to apply an RF voltage to the first andsecond AC/DC electrode stacks 320, 330, —for example by applyingopposite RF phases to successive ring or plate electrodes in the stacks.Both stacks could be thus united into a single stack. The RF electricfield produced by applying an RF potential to the stacks acts to preventions from landing on the electrodes and guide them through theseparation chamber 70′.

The power supply is also configure to apply a DC voltage to the stacks,for example by using a (resistive) potential divider connected to eachof the rings or plate electrodes in the stacks so as to permit a DCpotential gradient to be applied. As the ions enter into the separationchamber 70′, there is no gas pressure to propel them towards theaperture 75, so the DC gradient applied to the first and second AC/DCelectrodes 320,330 results in ions being pulled away from the separationchamber inlet 72.

The alternating phases of RF applied to the first and second AC/DCelectrodes 320, 330 are of a frequency and amplitude that results inwanted sample ions being guided along a path marked A′, away from theelectrodes and into the separation chamber outlet 75. Meanwhile unwantedsolvent and other ions are lost to the side walls of the separationchamber, because neutral solvent molecules experience no electric fieldand hence no accelerating or guiding force, and because any chargedsolvent ions (in particular) tend to aggregate as heavier clusters andare thus incapable of following the RF field. As may be seen in FIG. 3,the central axis and direction of travel of ions entering the separationchamber 70′ as a jet through the inlet 72, is generally at right anglesto the central axis and direction of travel of ions as they exit theseparation chamber 70′ through the outlet 75. This arrangement meansthat there is no direct line of sight between the inlet 72 and outlet75, preventing any uncharged solvent molecules, particulates and thelike from passing from the inlet 72 to the outlet 75 as a result solelyof an initial kinetic energy upon entering the separation chamber 70′.

It is preferable that the RF frequency applied to the first and secondAC/DC electrodes 320, 330 is in excess of 10% of the collision frequencyof the residual gas in the chamber, i.e., mainly, the residual gas fromthe desolvation region 60′, such as Nitrogen for example. It is alsopreferable that the RF amplitude be less than half of the breakdownvoltage of the residual gas at the chosen pressure of the separationchamber 70′.

As will be understood by the skilled person, the electrode arrangementin the separation chamber 70′ takes the form of an RF ion guide/massfilter, and it is thus desirable that the separation chamber beevacuated to a relatively low pressure, to reduce collisional losses.

The separation chamber 70′ is preferably evacuated to a pressure of nomore than around 5,000 Pa, but preferably to a pressure not lower thanaround 10 Pa using a pump (not shown in FIG. 3).

As with the arrangement of FIG. 2, it is desirable in the arrangement ofFIG. 3 that there is a pressure drop between the desolvation region 60′and the separation region 70′, so as to assist in the creation of a jetof ions and entrained solvent molecules as they enter the separationregion 70′. Although this is achieved in the specific configurationshown in FIG. 3 by evacuating the separation chamber 70′, so that thedesolvation region 60′ may remain at atmospheric pressure and hence notrequire enclosure/sealing, it will of course be understood that thedesolvation region 60′ could be enclosed so as to allow differentpressures (particularly, pressures above or below atmospheric pressure)to be set in the desolvation region 60′. As explained above, in order toemploy RF ion guide and/or mass filtering techniques in the separationchamber 70′, a relatively low pressure is desirable in that chamber(typically <50 mbar (5 kPa), preferably <0.2 mbar (20 Pa)). A quadrupolemass filter could be used in the separation chamber under such pressureconditions as a means to separate sample and solvent ions. Whilst, asdescribed, this can be achieved by retaining the desolvation region 60′at atmospheric pressure and then pumping the separation chamber 70′, byenclosing the desolvation region 60′, staged evacuation can be employed,wherein the liquid sample preparation region 10 is held at atmosphericpressure, whilst the desolvation region 60′ is roughly pumped to afraction of an atmosphere and the separation chamber 70′ is thenevacuated to a few thousand Pa, down to a few Pa or even lower.Enclosing the desolvation region 60′ also facilitates the use of aheated flow gas there, to assist in desolvation of the ionized eluent.The choice of whether the desolvation or separation chamber is sustainedat atmospheric pressure, is determined mainly by the properties of thereaction chamber. For example, in many cases it is beneficial to keep itat a pressure close to atmospheric pressure, in order to ensureefficiency of reaction. Atmospheric pressure also facilitates therequirement to move gases with appropriate velocities, and promotes easeof cleaning.

Sample ions exiting the separation chamber 70′ enter a reaction ordecomposition chamber such as a reaction chamber 80. The reactionchamber may, as with the arrangement of FIG. 2, be held at a pressureP_(oxid) higher than the pressure P_(separation) in the separationchamber 70′. This again permits the use of a counter gas flow from thereaction chamber 80 into the separation chamber 70′ via the separationchamber outlet 75, for the reasons previously explained. Ions could betransported against such flow using DC gradients for transport and RFfields for confinement.

Sample ions are then combusted in the reaction chamber 80. Optional CO₂separation may take place in a CO₂ separation unit 90, the sample ionflow may further optionally dried, and then isotopic ratio analysis maybe carried out by the IRMS 100 (FIG. 1).

The detection limit of the sample introduction system 50 described inFIGS. 1, 2 and 3 above is mainly defined by the number of ions necessaryto acquire a sufficient statistical accuracy of an isotopic ratio in theIRMS. As an example, for a typical current of 1 nA of a molecule with 20carbon atoms, about 10¹¹ molecules of CO₂ per second will be produced.At 100% ionisation efficiency, which is typical for high proton affinitymolecules, several picograms/second would be sufficient to deliver sucha current, with higher loads leading to saturation of the current.

The relatively low ionisation efficiency of the electron-impact ionsource in a standard IRMS (around 1 ion per 900 molecules) results in areduction by around 3 orders of magnitude, ie to around 10⁸ ions of CO₂per second. As a result, a statistically-limited accuracy of the isotoperatio for ¹³C/¹²C (with ¹³C at 1.1% of ¹²C) is around 0.1% rms over onesecond of acquisition. This is typically more than sufficient forroutine measurements in life science and (bio)-pharma applications, forlabelling experiments, etc. As the typical LC peak width is on the scaleof several seconds, online isotope ratio measurement becomes feasiblenotwithstanding possible peak tailing caused by combustion and CO₂separation.

To compensate for the low ionisation efficiency in IRMS, high currentsof sample ions are desired, up to microAmperes. This current (along witha high efficiency of ionisation) could be provided by an array of sprayprobes operating in parallel, each preferably spraying less than 1microliter/minute of eluent. A flat geometry of a heated channel 200 andseparation chamber 70 would support such parallel operation, with slitshaped separation chamber inlets 72 extending into the range of tens ofmm. Such larger inlets are capable of removing limitations caused byspace charge.

Although some specific embodiments have been described, it will beunderstood that these are merely for the purposes of exemplaryillustration of the invention and are not to be considered limitingthereof. Various modifications and additions may be contemplated. Forexample, although the embodiments of FIGS. 1, 2 and 3 all describe theuse of liquid samples with LC separation, ions might also be producedfrom a solid sample by techniques such as Matrix Assisted LaserDesorption Ionization (MALDI), direct electrospray ionization (DESI),direct analysis in real time (DART), etc.

Moreover, it is to be understood that the specific separation techniquesdescribed in connection with FIGS. 2 and 3 (using cross gas flow andcombined DC/DC/AC fields respectively) merely serve to exemplify thegeneral principles underlying the present invention. Generally speaking,it is necessary only that the separation chamber 70 be configured totransport wanted sample ions along an ion path across the chamber fromthe inlet 72 to the outlet 75 by the application of an electric field(AC and/or DC), whilst unwanted solvent ions and molecules are forced tomove along either another path that prevents them from passing throughthe outlet 75 and into the reaction chamber 80, or causes the unwantedsolvent ions/molecules to move indiscriminately in multiple directions.For example, separation of sample and solvent ions may be achievedthrough the use of a Field Asymmetric Ion Mobility (FAIMS) device. Sucha technique is shown in U.S. Pat. No. 6,690,004, which proposes a planarFAIMS arrangement, and in WO-A-00/08454 which shows a coaxialarrangement. In each case, sample and solvent ions are separated not ontheir mobility (which is linked to collision cross section and thusdirectly to m/z), but on differential mobility, which is more dependentupon the chemical structures of the respective ions.

According to the above, the separation chamber may therefore, as a meansto separate sample ions from interfering solvent ions and solventmolecules, comprise at least one of:

-   -   (i) an ion mobility separator (IMS), especially with transverse        gas flow, and/or preferably with off-set inlet and outlet,    -   (ii) an RF ion guide, optionally with DC axial field, having its        longitudinal axis different (preferably perpendicular) to the        axis along which ions enter the separation chamber, or having a        curved axis (curved away from the axis along which ions enter        the separation chamber).    -   (iii) a mass filter, for example a quadrupole mass filter,        optionally with a curved axis, or axis different to the axis        along which ions enter the separation chamber    -   (iv) an array of miniature (micrometer- or even nanometer-size)        mass filters arranged to divert ions away from neutral flow    -   (v) a Field Asymmetric Ion Mobility (FAIMS) device.

The arrangements of FIGS. 1 and 2 show, for the purposes ofillustration, a reaction chamber 80 connected to the separation chamber70. However it is to be understood that this is merely an example of asuitable decomposition or reaction chamber, and other techniques forgenerating products may be used.

FIGS. 4a and 4b show, respectively, schematic drawings of a part of thesample introduction system of FIGS. 1, 2 and 3, with first and secondalternative arrangements for reacting/combusting the ions arrivingthrough the separation chamber outlet 75. In order to avoid repetition,those features common to FIGS. 1-3 and FIGS. 4a and 4b , will not bedescribed in detail here. Moreover, the separation chamber in FIGS. 4aand 4b has been deliberately shown in highly schematic form, since theconcepts to be described below in respect of FIGS. 4a and 4b are equallyapplicable to either of the different specific separation chamberarrangements of FIGS. 2 and 3.

Turning first to FIG. 4a , ions pass through the desolvation region 60and into the separation chamber 70 through the inlet 72. Here, ions areseparated as previously described and ions of interest areguided/directed out of the separation chamber 70 via the outlet 75.

Upon exiting the separation chamber, ions pass along a conduit to afirst valve 400. The valve is switchable between a first position, inwhich ions arriving at it are directed along a first path into areaction chamber 80, and a second position in which ions arriving at thefirst valve 400 are directed along a second path and into a pyrolysis orreduction chamber 410. The valve may be either manually operated orunder software control so that, for example, a first set of ions may becombusted during a first period and then a second subsequent set of ionsmay be pyrolized during a second subsequent period (or vice versa).

Alternatively, the valve 400 may be configured to split the ion streamarriving at it so as to send part of the stream along the first paththrough the reaction chamber 80, whilst another part of the streamtravels along the second path through the pyrolysis chamber 410,simultaneously.

Following combustion or pyrolysis in the respective combustion orpyrolysis chamber 80, 410 respectively, the resultant (usually neutral)molecules or elements pass along further conduits and through a secondvalve 420 (either in series, if the first valve 400 is set to send ionseither to one or other of the combustion chamber 80 or pyrolysis chamber410, or in parallel if the ions are split so as to pass through both thecombustion chamber 80 or pyrolysis chamber 410 simultaneously), From thesecond valve 420, the products pass to the (optional) carbon dioxideseparation unit 90 (FIGS. 1-3) for onward reionization and massspectrometric analysis.

FIG. 4b shows an alternative configuration of a combustion chamber 80 inseries with the pyrolysis chamber 410, rather than parallel as in FIG.4a . In particular, in FIG. 4b , ions from the desolvation chamber 60pass through the separation chamber 70 and are separated there. Ions ofinterest pass through the outlet 75 and enter the combustion chamber 80.If it is desired to combust the ions, then this chamber is suitablyheated. Ions then exit the combustion chamber through an exit 415 intothe pyrolysis chamber 410. Where the ions have been combusted, thepyrolysis chamber 410 is not heated and simply acts as a conduit for thecombusted ions, which pass through the pyrolysis chamber 410, and outinto the optional CO₂ separation unit 90 (FIGS. 1-3) for subsequentanalysis.

Where on the other hand it is desired to pyrolize the ions, thecombustion chamber is instead not heated and simply guides incident ionsfrom the separation chamber 70 through the combustion chamber 80 andinto the pyrolysis chamber 410. The latter is heated so as to pyrolizethe ions before the resulting products are passed to the optional CO₂separation unit 90 (FIGS. 1-3) for subsequent analysis, instead.

As a further optional configuration, instead of simply directing theoutput of the sample introduction system 50 into the combustion chamber80 and/or pyrolysis chamber 410 and from there to an IRMS 100, forisotopic ratio measurements, a part of the resulting ions (such as aminor part, for example, around 10% or less) might be diverted to aconventional organic mass spectrometer, for carrying out analysis ofsample ions (MS) and/or their fragments (MS/MS; MSn). Suitableinstruments for such organic mass analysis are the triple quadrupole, orhigh resolution, accurate mass (HR-AM) devices such as the Exactive™ orQ Exactive™ instruments, manufactured by Thermo Fisher Scientific, Inc,which comprise an electrostatic orbital trap mass analyzer. Such anarrangement permits the analysis of isotopic ratios as well as molecularions and their fragments—and hence the molecular structure of the sampleions—in one workflow—potentially even in one dataset.

Also, more than one mass spectrometer could be used. For example, whilemost of ions (>90%) are transferred to combustion chamber and then toIRMS, remaining may be sampled into a conventional mass spectrometer,e.g. triple quadrupole, HR/AM instrument like Q Exactive (orbital trap),multi-reflection TOF, etc. In this way, both isotopic ratio andmolecular/structural information is obtained simultaneously andpossibly, in one data set.

One exemplary configuration to illustrate these concepts is shown inFIG. 5. Again this is a highly schematic diagram which (deliberately)does not specify the particular arrangement of the separation chamber70/70′. FIG. 5 shows a part of the system of FIG. 1. Ions from thedesolvation chamber 60 pass through the separation chamber where theyare separated as previously described. Ions of interest arrive at theoutlet 75 and then enter an ion storage device 500 where they may becooled and stored. A first set of ions may be ejected in a firstdirection—for example, axially as shown, where they enter a combustionchamber 80. From here, the ions are combusted and products enter theoptional CO₂ separation unit 90 (FIGS. 1-3) for subsequent analysis bythe IRMS 100 (FIG. 1).

A second set of ions held in the ion storage device 500 may instead beejected in a second direction—for example orthogonally—towards anorganic mass analyser 510 which in the example shown in FIG. 5 is anelectrostatic orbital trap mass analyser. A transient signal 520 isthereby obtained, from which a mass spectrum can be generated.

The ion storage device 500 may be any suitable device, such as a linearor 3D trap. To permit orthogonal ejection of ions stored in the ionstorage device 500 towards the electrostatic orbital trap mass analyser510 shown in FIG. 5, the ion storage device 500 might for example be acurved linear trap.

By storing the ions passing through the outlet 75 in the separationdevice 70 in an ion storage device 500, those ions selected to beanalysed by the organic mass analyser 510 may be ejected directlythereto without further treatment. Meanwhile, ions to be combusted passthrough the reaction chamber 80. The resultant products subsequentlythen require further ionization using the second ionization source 110(FIG. 1). A technique has been described in which ions enter the ionstorage device 500 and are then directed in different ways, dependingupon what is to be done with them (combustion or otherwise). However,the ions introduced into the ion storage device 500 need not be of asingle species (or single, fixed range of species) during all suchanalyses. The ions can instead be selected in accordance with theirsubsequent treatment. For example, during a first period, ions of afirst species (or range of species) may be selected, by appropriateconfiguration of the electric fields in the separation chamber 70/70′,may enter the ion storage device 500, and then those ions may be ejectedto the reaction chamber 80 for subsequent combustion and analysis by theIRMS 100. In a second time period, the electric field in the separationchamber 70/70′ may instead be configured to select ions of a secondspecies (or second range of species), different to the first, which arethen trapped in the ion storage device 500 and instead ejected to theorganic mass analyser 510 for analysis there.

Of course, the configurations of FIGS. 4a, 4b and 5 are entirelycompatible: in other words, instead of simply ejecting ions from the ionstorage device 500 into a reaction chamber 80, they could instead beejected to a serially configured combustion and pyrolysis chambers (FIG.4b ) or a parallel arrangement with valves (FIG. 4a ).

One potential practical implementation of the liquid sample preparationregion and sample introduction system described above may be achieved bymodification of the Q Exactive hybrid quadrupole-Orbitrap massspectrometer manufactured by Thermo Fisher Scientific, Inc. Thearrangement of components is shown schematically in, for examplehttp://planeorbitrap.com/g-exactive. In the Q-Exactive massspectrometer, ions are typically generated by an atmospheric pressureelectrospray (ESI) source, and then injected into a first stage of theapparatus. This first stage may be configured to act as the desolvationchamber 60 of earlier Figures. It has a heated channel which may be usedas the heated channel 200 of FIG. 2, for example.

Downstream of the first stage acting as a desolvation chamber 60, is abent multipole ion guide which may remove neutral ions whilsttransmitting charged analyte particles of interest. After that is aquadrupole mass filter which can be configured as a separation chamber70. Finally, the Q-Exactive device comprises an Orbitrap massspectrometer. This may be employed if the arrangement of FIG. 5 above isdesired. A suitable reaction chamber may then be added to the back endof the Q-Exactive device.

In order to achieve effective oxidation, it is desirable that relativelyhigh pressures are employed (in particular, many Pa). It is thereforepreferable to use just the first one or two pumping stages of theQ-Exactive interface. It may also be necessary subsequently to increasethe pressure again.

1. A method of introducing a sample into an Isotope Ratio Spectrometer,comprising: (a) generating sample ions in a solvent matrix; (b) removingat least a proportion of the solvent matrix from the sample ions in adesolvation chamber, so as to produce a flow of sample ions along withnon-ionised solvent and solvent ions; (c) in a separation chamber havinga pressure, P_(sep), lower than a pressure, P_(sampling), in thedesolvation chamber, applying an electric field to the flow of ionsalong with solvent vapours, so as to direct wanted sample ions towardsan outlet of the separation chamber, whilst unwanted solvent ions andnon-ionised solvent are directed away from the said separation chamberoutlet; and (d) decomposing the sample ions to molecular products oncethey have passed through the outlet of the separation chamber.
 2. Themethod of claim 1, wherein the pressure, P_(sep), within the separationchamber is substantially atmospheric pressure.
 3. The method of claim 2,further comprising controlling the pressure within the desolvationchamber to be at least 200-300 kPa.
 4. The method of claim 1, furthercomprising controlling the pressure, P_(sampling), within thedesolvation chamber, so as to be at least twice the pressure P_(sep) inthe separation chamber.
 5. The method of claim 1, wherein the pressurewithin the desolvation chamber is substantially atmospheric pressure,the method further comprising controlling the pressure within theseparation chamber to be at no greater than 10-30 kPa.
 6. The method ofclaim 1, wherein the step (b) further comprises directing the flow ofions and non-ionised solvent vapours to a separation chamber inlet, sothat the ions and solvent vapours enter the separation chamber throughthe separation chamber inlet in a first direction defining a first axis,the method further comprising directing the wanted sample ions exittoward the separation chamber outlet so that they exit the separationchamber in a second direction defining a second axis, and wherein thefirst and second axes are not coincident.
 7. The method of claim 6,wherein the step (c) further comprises supplying a flow of dry gas in adirection transverse or counter to the said first axis so at to separateions within the separation chamber in accordance with their mobility. 8.The method of claim 6, further comprising generating an AC and/or a DCelectric field within the separation chamber, so as to cause the wantedsample ions, having a first mass to charge ratio or range of mass tocharge ratios, to be directed along a first flow path towards theseparation chamber outlet, but to cause the unwanted solvent ions,having a second mass to charge ratio or range of mass to charge ratios,different to the said first mass to charge ratio or range of ratios, tobe directed away from the separation chamber outlet.
 9. The method ofclaim 1, further comprising supplying a counter gas to the separationchamber outlet in a direction generally opposed to the direction ofincidence of the sample ions thereat.
 10. The method of claim 1, whereinthe step (d) of decomposing the sample ions comprises pyrolizing oroxidising or reducing the sample ions.